ИСТИНА

Chromatin organization still remains a challenging question despite numerous researches that give rise to contradictory models varying from “polymer melt” to hierarchical chromatin folding models. Such controversy is mainly caused by differences in methodology applied where preservation of native chromatin structure presents one of the most significant issues. Since chromatin higher-order structures extend from 10nm nucleosome fiber to giant chromosomes a broad range of techniques is required for their visualization. Developing new labeling and imaging approaches represents a promising way to reveal the hidden structure of chromatin. To visualize a higher-order chromatin structure at high resolution in non-destructive way we combined structural illumination microscopy and immunoelectron microscopy. We applied replicative labeling of DNA by “click” chemistry to maximally preserve chromatin structure, with subsequent Nanogold-coupled antibodies and silver enhancement to specifically label a subset of chromatin domains with distinct functional state and compaction level. Labeling of the same molecules for light and electron microscopy avoids any problems caused by non-perfect matching of optical and physical slices, any distortions resulting from epoxy embedding procedure and sectioning. Collection of information about structure at SIM-level allows highly efficient analysis of large field of views, making possible live imaging. TEM overcomes restrictions of light microscopy resolution and provides rich structural context of the cell. Thus appropriate SIM-TEM correlation displays advantages of both approaches with increased throughput while overcoming disadvantages of both methods. This simple and highly sensitive approach allows tagging specific chromatin fractions according to its replication timing and makes it possible to effectively study chromatin organization and its post-replicative dynamics.